Senior Honors Projects, 2010-current

Date of Award

Spring 2015

Document Type

Dissertation/Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Chemistry and Biochemistry

Advisor(s)

Gina MacDonald

Abstract

The Escherichia coli protein RecA catalyzes the strand exchange reaction used in DNA repair and genetic recombination. RecA is also a target for inhibiting microbial antibiotic resistance, understanding cancer propagation, and characterizing neurodegenerative disorders. Therefore, understanding factors that affect RecA structure and stability is broadly applicable to many fields. Previous studies in our lab have shown buffer-specific changes in RecA stability and unfolding transitions. These studies suggest only minimal buffer-dependent changes in nucleotide binding and secondary structure but do not explain the significant differences in RecA stability and unfolding profiles. Here we have employed various biochemical and spectroscopic techniques to further characterize RecA both structurally and functionally in four common buffers: Tris, MES, HEPES, and Phosphate. Activity assays reveal RecA activity may be slightly decreased in HEPES and Phosphate buffers as compared to Tris and MES. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy show conservation of global RecA secondary structure in all buffers, yet unique aggregation states are observable in CD turbidity plots. Laser-induced photolysis of caged nucleotides was also used in conjunction with difference FTIR spectroscopy to generate RecA-ADP minus RecA difference infrared spectra in each of the four buffers. These studies detected unique buffer-specific changes in nucleotide binding to RecA in each buffer and may provide insight into mechanisms for buffer-specific stability profiles of RecA.

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